Recovery and purification of therapeutic proteins accounts for approximately 75% of the manufacturing costs of biological drugs. Improving the efficiency and/or reducing cost in the purification step is of general interest. The general industrial purification process often includes a number of unit operation steps, like extraction, precipitation, as well as anion- and cation-exchange chromatography. Affinity chromatography is the preferred downstream process step due to its high recovery, yield and specificity, but the current cost and limitations of affinity chromatography is very substantial and in many cases prohibitive for a more general and efficient use of this unit operation.
Conventional affinity chromatography is in general characterized by having a capturing ligand immobilised to a solid phase matrix. The ligand reversibly binds a target molecule present in a fluid such as liquid culture medium or serum. Target molecules are recovered by dissociating the complex at eluting conditions. Commercially available affinity matrices are in a ready to use format including capturing ligands covalently attached to the matrices. In conventional affinity chromatography the dissociation constant, KD, between the ligand and the target protein is in the range of about 10−5-10−7M. Interactions with dissociation constants exceeding 10−10-10−11M are often impossible to use, as the conditions required to dissociate the complex are then the same as those that will result in denaturation of the target proteins.
In the co-pending application WO 2009062942 we have previously demonstrated the feasibility of applying a generic capturing ligand immobilised to a matrix, and a semi generic dual affinity polypeptide with different binding affinity toward the target biomolecule and the capturing ligand respectively in a purification process. The dual affinity polypeptide (DAP) reacts with the target biomolecule to form a 1:1 complex of medium binding affinity in which one DAP binds to one target, and this complex subsequently binds non-covalently to a generic affinity matrix with a strong binding affinity. The target molecule is recovered by specific elution from the generic matrix leaving the dual affinity polypeptide attached to the capturing ligand on the matrix, due to the tight binding to the ligand preventing leakage from the solid phase matrix.
It is the goal of the present invention to further improve this system by increasing the binding capacity of the DAP molecule.